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Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector.

ABSTRACT: Since melanomas often retain wild type p53, we developed an adenoviral vector, AdRGD-PG, which provides robust transduction and transgene expression in response to p53. Previously, this vector was used for interferon-? gene transfer in mouse models of melanoma, resulting in control of tumor progression, but limited cell killing. Here, the AdRGD-PG-hIFN? vector encoding the human interferon-? cDNA (hIFN?) was used to transduce human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild type p53). In vitro, cell death was induced in more than 80% of the cells and correlated with elevated annexinV staining and caspase 3/7 activity. Treatment with hIFN? promoted cell killing in neighboring, non-transduced cells, thus revealing a bystander effect. In situ gene therapy resulted in complete inhibition of tumor progression for SK-MEL-147 when using nude mice with no evidence of hepatotoxicity. However, the response in Nod-Scid mice was less robust. For SK-MEL-05, tumor inhibition was similar in nude and Nod-Scid mice and was less efficient than seen for SK-MEL-147, indicating both cell type and host specific responses. The AdRGD-PG-hIFN? vector provides extensive killing of human melanoma cells in vitro and a potent anti-tumor effect in vivo. This study provides a critical advance in the development of our melanoma gene therapy approach.

SUBMITTER: David TIP

PROVIDER: S-EPMC7578831 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector.

David Taynah I P TIP Cerqueira Otto L D OLD Lana Marlous G MG Medrano Ruan F V RFV Hunger Aline A Strauss Bryan E BE

Scientific reports 20201021 1

Since melanomas often retain wild type p53, we developed an adenoviral vector, AdRGD-PG, which provides robust transduction and transgene expression in response to p53. Previously, this vector was used for interferon-β gene transfer in mouse models of melanoma, resulting in control of tumor progression, but limited cell killing. Here, the AdRGD-PG-hIFNβ vector encoding the human interferon-β cDNA (hIFNβ) was used to transduce human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild type p53 ...[more]

PMID: 33087767

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Project description:The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.A synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.Transfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.The TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues.

Dataset Information

Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector.

Publications

Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector.

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